The present invention relates to a method for screening drugs for use in treating hypertension using the tubular renin-angiotensinogen system identified by the present invention. The invention further relates to a method to diagnose sodium status in an individual by measuring urinary angiotensinogen, angiotensin-I, des-AI-angiotensinogen or renin.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended list of references.
The following abbreviations are used herein: A-I-angiotensin-I; A-II-angiotensin-II; ACE-angiotensin converting enzyme; AGTxe2x80x94angiotensinogen gene; ANG or Angxe2x80x94angiotensinogen protein; -6(A)/-6(G)xe2x80x94promoter polymorphism at position -6; CCDxe2x80x94cortical collecting duct; CNTxe2x80x94cortical connecting tubule; DCTxe2x80x94distal convoluted tubule; ICxe2x80x94intercalated cells; JGAxe2x80x94juxtaglomerular apparatus; PCRxe2x80x94polymerase chain reaction; RASxe2x80x94renin-angiotensin system; RT-PCRxe2x80x94reverse transcriptase polymerase chain reaction; and HPLCxe2x80x94high pressure liquid chromatography.
Blood pressure control is intrinsically linked to fluid volume balance and electrolyte homeostasis. Regulation of plasma volume in response to variation in dietary sodium (1) is primarily controlled by the renin-angiotensin system (RAS) and its main effector angiotensin-II (A-II); this peptide hormone is released from angiotensinogen (Ang) by two cleavage steps involving renin and angiotensin-converting enzyme (ACE) (2).
The short-term effects of A-II are better understood than its long-term effects. Acute depletion of body fluid volume triggers a vasoconstrictor response mediated by the circulating renin-angiotensin system (RAS), involving renin secreted by the juxtaglomerular apparatus (JGA) in the kidney, Ang from liver, and ACE present in the luminal cell membrane of capillary endothelium.
Sustained low-dose infusion of A-II leads to progressive, long-term elevation of arterial pressure due to cumulative sodium retention primarily mediated by direct intrarenal A-II effects 1. A-II has been detected in proximal tubular luminal fluid at high concentrations (3, 4). In contrast to plasma renin (36-40 kDa), Ang (61-65 kDa) is not filtered through the glomerular basement membrane. Detection of abundant angiotensinogen mRNA in proximal tubule epithelium (5-7), strongly suggests local generation of A-II at this site by an as yet unspecified mechanism. Renin mRNA can be detected in proximal tubule only by application of the very sensitive technique of RT-PCR (8). Exogenous A-II stimulates the luminal sodium-hydrogen exchanger present in the proximal tubule cells (9, 10) and also stimulates epithelial sodium channels and possibly other transporters in the distal segments of nephron (11-14).
Fundamental questions remain unanswered, however. If intrarenal A-II directly affects sodium reabsorption, where is it generated, and by what mechanism? How is this mechanism regulated in response to sodium? At what sites does A-II impact on sodium transport along the nephron? What is the mechanism for coordinated regulation of sodium uptake in proximal and distal segments of the nephron? Can it allow for a decoupling of sodium reabsorption and potassium excretion in the distal tubule?
It is desired to address these questions and to elucidate answers which can be used for screening drugs and diagnosing sodium status of an individual.
In accordance with the present invention, it is shown that proximal tubule cells cultured as a polarized monolayer secrete Ang at their apical side, and that Ang transits through the entire nephron as it can be measured in final urine. Furthermore, it is shown that renin, in addition to being filtered, is expressed in a specific segment of the nephron, the connecting tubule. Furthermore, angiotensinogen expression in proximal tubules and renin expression in connecting segments (distal arcades) is an inverse function of dietary sodium.
The data disclosed herein suggest that filtered renin, Ang secreted into proximal tubule, and renin in connecting tubule, together with luminal angiotensin-converting enzyme (ACE) and A-II receptors, previously demonstrated in the luminal fluid and the apical cellular membranes of proximal tubules and collecting ducts (15, 16), define a tubular RAS involved in the control of sodium reabsorption as a function of dietary salt. This tubular RAS could contribute to body fluid control and blood pressure regulation. Furthermore, genetic differences in the angiotensinogen gene (17, 18) may influence susceptibility to essential hypertension through their impact on this tubular system.
Thus, the present invention relates to a method for screening drugs for use in treating hypertension using the tubular renin-angiotensinogen system identified by the present invention. The invention further relates to a method to diagnosis sodium status in an individual by measuring urinary angiotensinogen, angiotensin-I, des-AI-angiotensinogen or renin.
It has been discovered that angiotensinogen, its enzyme catalyzed products or renin excreted in urine vary with changes in dietary sodium. Thus, the sodium status of an individual is diagnosed by determining the amount of angiotensinogen or its enzyme catalyzed products or renin in the urine of the individual and comparing the determined amount with normal values. A finding of elevated levels of these compounds indicates high sodium. An individual""s sodium sensitivity can also be determined by determining the amounts of these compounds in urine. Levels of these compounds are determined using conventional techniques, and any appropriate method is suitable for use. If the levels are elevated in an individual under a high salt diet compared to reference values, the individual is sensitive to salt.
It has also been found that the expression of angiotensinogen and renin is regulated at specific sites along the kidney. This finding identifies new therapeutic targets for the blood pressure control and provides the basis for a method to screen drugs for use in treating hypertension. According to the present invention, drug candidates for treating hypertension are screened using the tubular renin-angiotensin system by testing the effects of drug candidates at the proximal and/or distal tubule.